Cotton is a globally important crop, grown primarily for fiber. Seeds provide an important source of food for livestock. Cotton has influenced economic development of many nations, throughout the world. Therefore, cotton improvement programmes by modern methods of agrobiotechnology are of interest worldwide. This has increased the importance of developing tissue culture methods to facilitate the application of modern techniques of genetic engineering of cotton plant.
Several reports on tissue culture of cotton have been published. These are related to direct shoot differentiation and somatic embryogenesis through suspension and callus cultures. These are listed below by way of references.
Organogenesis and regeneration, leading to micropropagation by tissue culture methods have been successfully demonstrated in several plant species e.g. Phaseolus sp. (Rubluo A & Kartha K K 1985). In vitro culture of shoot apical meristems of various Phaseolus species and cultivars, J. of Plant Physiol 119: 425-433. Glycine max (Shetty K., Asano Y and Oosawa K 1992 Stimulation of in vitro shoot organogenesis in Glycine max Merrill by allantoin and amides. Plant Sci. 81:245-252). Cajanus cajan(Shiv Prakash N, Pental D & Bhalla-Sarin N 1994 Regeneration of Pidgeonpea (Cajanus cajan) from cotyledonary node via multiple shoot formation. Plant Cell Rep. 13:623-622), Carnation (Claire Annex A. Yancheva S and Dons H 1995. Cells within the nodal region of carnation shoots exhibit a high potential for adventitious shoot formation. (Plant Cell Tiss. Org. Cult. 40:151-157) etc.
Plant regeneration by tissue culture techniques is well established. A wide variety of plant species has been successfully regenerated in vitro via organogenesis or somatic embryogenesis. Organogenesis leads to organ formation i.e., shoot (or root), which can be isolated to induce development of roots (or shoots) to produce full plant while somatic embryogenesis leads to the development of somatic embryos (embryos developed without fertilization) which have both shoot and root initials and are capable of developing into whole plant. Although the ability of individual parts of plants and cells to regenerate into complete plants (called totipotency) is a well known phenomenon, each plant or plant part requires specialized studies to invent the conditions that allow such regeneration. Some of the broadly applicable factors controlling growth and differentiation of such cultures have been determined. The establishment of interactions among different groups of phytohormones and growth regulators alone or in combinations are responsible for certain interrelations existing among cells, tissues and organs. There seems to be consensus that the success in inducing differentiation depends upon the type of explant, physiological condition of the explant and physical and chemical milieu of the explant during culture. Due to this, the science of tissue culture has been directed to optimize the physiological conditions of source plant, the type of explant, the culture conditions and the phytohormones used to initiate tissue culture. This substantiates the fact that development of a new process for proliferation of plants by tissue culture is not obvious.
One major aspect that has to be investigated on case to case basis is the type of plant growth regulators and the amount of plant growth regulators that induce regeneration. Besides chemical composition of the medium, temperature of growth and other culture conditions play important role in the induction of organogenesis and somatic embryogenesis and maturation of shoots and roots and the formation of healthy fertile plants. The response to medium, hormones and growth conditions differs from plant species to species and variety to variety. Thus, inventing conditions for efficient regeneration of plants, organogenesis and somatic embryogenesis requires developing specialized knowledge about a given plant.
Another major area where innovativeness is required in tissue culture is identifying the plant part that efficiently responds to the culture conditions and leads to prolific regeneration. Not all plant parts of a given species are amenable to efficient regeneration. It is a complex combination of the plant part identified for totipotency (called explant), the physiological state of the explant and the growth conditions, especially the growth regulators that determine success of a plant in tissue culture. Different explants from a given plant usually show very different response to growth conditions for proliferation. No general principles can be applied to achieve regeneration. In each case, identification of the explant and identification of the culture conditions are innovative steps in the development of a tissue culture method for regeneration of a plant part into a number of plants or somatic embryos.
As of now, a detailed publication on the formation of multiple shoots from any tissue explant of cultivated varieties of cotton is not available. This invention describes for the first time a detailed protocol for organogenesis from a small part (called an explant) of cotton seedling to give multiple shoots of cotton plant through tissue culture. The method is very useful in agricultural biotechnology for micropropagation and genetic transformation because it shows wide applicability to all the cultivars tested by the inventors and the shoots can be efficiently raised to maturity. The method has a great potential in cotton improvement programmes by modern methods of agrobiotechnology.
Several reports deal with tissue culture conditions (Davidonis G H and Hamilton R H, 1983 Plant regeneration from callus tissue of Gossypium hirsutum L. Plant Sci. Lett. 32: 89-93; Shoemaker R C, Couche L J & Galbraith D W 1986). Characterization of somatic embryogenesis and plant regeneration in cotton (Gossipium hirsutum L) Plant Cell Rep. 3:178-181; Trolinder N L and Goodin J R 1987; Somatic embryogenesis and plant regeneration in cotton Gossipium hirsutum L. Plant Cell Rep. 6:231-234 Finer J. 1988). Plant regeneration from somatic embryogenesis suspension cultures of cotton Gossipium hirsutum. (Plant Cell Rep. 7:399-402) that give rise to somatic embryos (structures that give rise to normal plants without going through fertilization) have been reported. Initiation and maturation of somatic embryos take several months. The method is highly dependent on genotypes (Trolinder N L and Xhixian C., 1989 Genotype specificity of the somatic embryogenesis response in cotton. Plant Cell Rep. 8:133-136) and is, therefore, applicable to a restricted group of varieties. Initiation and maturation of somatic embryos takes several months, and the plants regenerated via somatic embryogenesis were of ten reported to be cytologically and morphologically abnormal (Stelly D M, Altman D W, Kohel Rz, Rangan T S & Commeskey E 1989. Cytogenetic abnormalities of cotton somaclones from callus cultures. (Genome 32:762-770). Plants developed via somatic embryogenesis were also of ten reported to be sterile (Trolinder N L & Goodin J R 1987, Somatic embryogenesis and plant regeneration in cotton Gossipium hirsutum L. Plant Cell Rep. 6:231-234).
These regeneration processes have been successfully used in Agrobacterium mediated gene transfer in cotton (Umbeck P, Johnson G, Barton K. Swain W 1987 "Genetically transformed cotton Gossipium hirsutum L. Plant Bio./ Technology 5:263-266; Firoozabady E., DeBoer L D, Merlo J D, Halk L E, Amerson, N L, Rashka K E and Murrey E E 1987. Transformation of cotton Gossypium hirsutum L. by Agrobacterium tumefaciens and regeneration of transgenic plants. Plant Mol. Biol. 10:105116) and in particle bombardment procedure (Finer J J and McMullen M 1990, Transformation of cotton Gossypium hirsutum L. via particle bombardment Plant Cell Rep. 8:586-589). Certain bacterial genes, like those encoding herbicide resistance (Bayley C, Trolinder N L, Ray C, Morgan M, Quisenberry J E and OW DW 1992, Engineering 2.4-D resistance into cotton (Theo. Appl. Genet. 83:45-649) and Bacillus thuringiensis endotoxin genes (Perlak F J, Deaton R W, Armstrong T A, Fuchs R L Sims S R, Greenplate J T & Fischhoff D A 1990. Insect resistant cotton plants. Bio/Technology 8:939-943) have been successfully expressed in transgenic cotton plants. But these advances are presently restricted only to Coker cultivars of cotton because other cultivars did not respond to the above mentioned tissue culture i.e., somatic embryogenesis protocols.
There have been publications describing in vitro development of a single shoot from a shoot apex tissue of cotton plant under aseptic tissue culture conditions (Gould J., Banister S, Hassegawa O, Fahima M, Smith R H 1991. Regeneration of Gossypium hirsutum and G. barbadense from shoot apex tissue for transformation. Plant Cell Rep. 10:12-16). Apical meristems and shoot tips of cotton plants have been used for culture to obtain callus, adventitious buds and multiple shoots (Bajaj Y P S and Manjeet Gill 1986: Preservation of cotton Gossypium sps. through shoot tip and meristem culture (Indian J. of Exp. Biol. 24:581-583). In these reports, the response of the cultures was genotype dependent and rooting was very infrequent. Due to this problem, the reproducibility of these processes was not very good.
Recently, (McCabe D E and Martinelli B J 1993, Transformation of elite cotton cultivars via particle bombardment of meristems Bio/Technology 11:596-598; Chlan C A, Lin J, Cary J W & Cleveland T E 1995, A procedure for biolistic transformation and regeneration of transgenic cotton from meristematic tissue (Plant Mol. Bio. Rep. 13:31-37) cotton embryonic axes were used for introducing genes by biolistic transformation method. In these reports, a single apical meristem grew into a single shoot. The regenerated shoots were then rooted on suitable medium to obtain mature plants of cotton. In all such reports, transformation and regeneration were inefficient since a single apical meristem developed into a single mature plant and most of the transformants were chimeric with respect to the expression of transformed genes. Such process can be largely improved by developing a process wherein many plants develop from single explant, as disclosed in this invention.
Table 1 summarises the state of art of tissue culture process related to cotton plant as covered by patents or described in literature. It is then followed by a statement describing the process invented by us in contrast to the known state of art.
The abbreviations used in the text for the plant growth regulators (hormones) employed in the culture medium are: BAP (6-benzyl amino purine or 6-benzyl adenine), 2iP (.gamma..gamma.dimethyl allylamino purine), Kin (kinetin), IAA (indole acetic acid), NAA (naphthalene acetic acid), IBA (indole butyric acid), TDZ (1-phenyl-3, 1.2.3 thidiazol-5-yl urea), DU (diphenyl urea), PU (U-1-phenyl N 4 pyridyl urea) and 2.4D (2.4 diphenoxy acetic acid).
TABLE 1 State of art for tissue culture regeneration in cotton. Report Mode of regeneration Phytohormones Explant Remarks 1. Davidonis GH and Hamilton RH Somatic NAA and Cotyledon 2 year old calli of G. hirsutum L. cv. (1983) Plant Regeneration from embryogenesis Kinetin Coker 310 grown on LS medium callus tissue of G. hirsutum L. containing 30 gm/L glucose in Plant Sci. Lett. 32:89-93 abscence of NAA and kin were used. 30% cultures gave rise to somatic embryos. 2. Davidonis GH, Mumma RO, --do-- --do-- --do-- --do-- Hamilton RH, 1987 controlled regeneration of cotton plants from tissue culture U.S. Pat. No. 4672035 3. Shoemaker RC, Couche LS, and Somatic NAA, Kinetin Hypocotyl 17 cultivars of cotton G. hirsutum Galbraith DW 1986, embryogenesis L. were evaluated for somatic characterization of somatic embryogenesis. After a series of embryogenesis and plant transfer of calli through medium regeneration in cotton Gossypium containing MS salts, NAA & kin. hirsutum L. Plant Cell Rep. After several weeks calli were 3:178-181 observed for the presence of somatic embryos. Cultivars Coker 201 and Coker 315 were identified as embryogenic. The embryos were isolated and developed into plants. 4. Trolinder NL, and Goodin JR Somatic 2,4-D, Kin Hypocotyl Globular Embryos were observed in 1987, somatic embryogenesis and embryogenesis 6-week-old callus culture. At this Plant regeneration in cotton stage calli were subcultured to liquid Gossypium hirsutum L. Plant Cell suspension in growth regulator free Rep 6:231-234 medium. After 3-4 weeks suspensions were sieved to collect globular and heart stage embryos. Collected embryos were developed on solidified medium to maturity. Mature embryos were germinated into plants. Most of the plants developed by this method were sterile (only 15% of the plants were fertile). 5. Trolinder NL and Xhixian C Somatic 2,4-D & Kin Hypocotyl 38 cultivars, strains and races of 1989, Genotype specificity of the embryogenesis Gossypium were screened for somatic embryogenesis response somatic embryogenesis with the in cotton. Plant Cell Rep. 8:133- method developed for Coker 312. 136 Screening indicated that genotype variation for embryogenesis existed. Only a few genotype are amenable to the model developed for Coker 312. 6. Stelly DM, Altman DW, Kohel A high frequency of chromosomal RZ, Rangan TS and Commeskey anomaly was observed in plants E 1989, cytogenetic regenerated through somatic abnormalities of cotton cultures. embryogenesis and most of the Genome 32:762-770 plants regenerated through somatic embryogenesis were sterile. 7. Finer J 1988, Plant Regeneration Somatic NAA, Kin Cotyledon Maintainable embryogenic from somatic embryogenesis embryogensis Picloram,2,4-D suspension cultures were developed. suspension cultures of cotton Embryos were developed by Gossypium hirsutum Plant Cell transferring embryogeneic tissue to Rep 7:399-402. auxin free medium. Plants derived were fertile. 8. Finer J, 1990. An efficient --do-- --do-- --do-- --do-- method for regenerating cotton from cultured cells. Patent No. ZA/A8808599 9. Rangan TS: 1993 Regeneration Somatic NAA, Kin Hypocotyl, Callus was initiated on MS medium of cotton Patent No. 5244802 embryogenesis Cotyledon, containing NAA & kin, subcultured Immature every 3.sup.rd week for growth. Somatic embryo embryos were formed four to six months after placing tissue on callus initiation medium. Many varieties were identified as embryogenic in vitro are SJ2, SJ4, SJ5, SJ2C, GC510, B1644, B2710, Siokra and FC 2017. 10. Gawel NJ and Robacker CD Somatic 2,4D & kin Petiole from A comparative study was made for 1990 somatic embryogenesis in embryogenesis mature somatic embryogenesis in liquid vs two Gossypium hirsutum flowering plants solid media & it was found that genotypes on semisolid vs liquid culture on liquid media favors the proliferation media. Plant Cell somatic embryogenesis in both the Tissue Organ Culture 23:201-204 genotypes named Coker 312 & T- 25. 11. Bajaj YPS and Gill M, 1986 Callus culture NAA, IAA, kin Shoot tip Response of culture was genotype Preservation of cotton Adventitious bud dependent and only a few cultures Gossypium sps through shoot tip culture & multiple gave multiple shoots while rooting and meristem culture. Indian J. of shoot was also very infrequent. Exp. Biol. 24:581-583 12. Gould J, Banister S, Hassegawa Organogenesis from Nil Apical shoot Normal and fertile plants of G. O, Fahima M., Smith RH 1991. pre-existing meristems meristem barbedense Pima S-6 and 19 Regeneration of Gossypium cultivars of G. hirsutum were hirsutum and G. barbedense from regenerated using this method but shoot apex tissue for only one shoot is formed from one transformation. Plant Cell Rep. explant & rooting could not be 10:12-16 optimised. 13. Umback P, Johnson G, Barton K, Genetic 2,4-D,Kinetin Hypocotyl Method of genetic transformation of Swain W, 1987 genetically transformation of cotton was disclosed. Immature transformed cotton (Gossypium cotton & regeneration tissue of cotton was transformed in hirsutum L). Plant. of plants vitro by Agrobacterium tumefaciens Biotechnology 5:263-266 mediated genetic transformation method. The resulting cotton tissues were screened for transformation by selection on drug. Transformed cultures were then induced to give somatic embryos. The somatic embryos were developed into mature plants. Coker 310, 312 & 5110 were transformed by this method. 14. Umback P, 1991 Method of producing transformed cotton cells by tissue culture, Patent No. IN A 168950 15. Firoozabady E. DeBoer LD, Genetic 2iP, NAA Cotyledon Cotyledon explants from 12 day old Merlo JD, Halk LE, Amerson Transformation & seedlings were transformed and NL, Rashka KE and Murrey EE regeneration of plants were regenerated. For 1987, Transformation of cotton transgenic plants regeneration the explant treated with Gossypium hirsutum L. by Agrobacterium tumefacines were Agrobacterium tumefaciens and transferred to medium containing regeneration of transgenic plants. bacteriostatic & selective agents so Plant Mol. Bio. 10:105-116 that only transformed cells give rise to callus. Callus when subcultured to hormone free medium gave rise to transgenic plants. Using this method G. hirsutum Coker 201 was transformed. 16. Perlak FJ, Deaton RW, Transformation & 2,4-D, Kinetin Hypocotyl Truncated forms of the insect control Armstrong TA, Fuchs RL, Sims regeneration of protein genes of Bacillus thuringiensis SR, Greenplate & Fischoff DA Transgenic Plants (via var. Kurstaki HD-1 (cry 1A(b) and 1990. Insect resistant cotton somatic HD73 CrylA(c)) were transformed into Plants Bio/Technology 8:939-943 embryogenesis cotton hypocotyl section via Agrobacterium tumefaciens and somatic embryos were obtained from transformed cells and finally insect resistant cotton plants of G. hirsutum cv. Coker 312 were obtained. 17. Baylay C, Trolinder NL, Ray C, Transformation & 2,4-D, Kinetin Hypocotyl 2,4-D monooxygenase gene tfd A Morgan M, Quisenberry JE and regeneration (via from Alcaligenus eutrophus was OwDW 1992. Engineering 2,4-D somatic isolated, modified and expressed in resistance into cotton. Theo. embryogenesis of tobacco and cotton plants Appl. Genet. 83:645-649 transgenetic plants) transformation was done by Agrobacterium tumefaciens and 2,4D resistant plants of Coker 312 line were obtained. 18. Rangan T, Rajsekran, Hudspeth Transformation and NAA, Kinetin Hypocotyl, Regeneration and transformation of and Yenofsky (1989) regeneration (via cotyledon, cotton (G. hirsutum var. Acala SJ2, Regeneration and transformation somatic Immature SJ4, SJ5, SJ2-1, GC510, B1644, of cotton Patent No. EP344302 embryogenesis) of embryo B2724, B1810, Picker Variety of transgenic plants. Siokra & stipper var FC2017) transformation was done by Agrobacterium tumefaciens somatic embryos obtained from callus were germinated on Beasley and Ting's medium. 19. Finner JJ and Mc Mullen (1990) Transformation & NAA, kin, Cotyledonary Embryogenic suspension cultures of Transformation of cotton regeneration (via 2,4D leaf disc cotton were subjected to particle Gossypium hirsutum L. via somatic picloram bombardment where high density particle bombardment Plant Cell embryogenesis) of particles carrying plasmid DNA Rep. 8:586-589 transgenic plants were accelerated towards the embryogenic plant cell. These cells were then subjected to developmental process of somatic embryo in the presence of selection agent (Hygromycin) cv. Coker 310 was transformed by this method. 20. McCabe DE and Martinelli BJ Organogenesis from BA (a brief embryonic axes The process involves excising the (1993) Transformation of Elite pre-existing meristems treatment for embryonic axes from germinating cotton cultivars via particle 15 hrs) seeds and blasting particles carrying bombardment of meristems Bio/ foreign genes into the embryonic Technology 11:596-598 axes. From the treated embryonic axes, plants were developed and screening was done for transformed plants. Only one plant develop from a single axes. Using this method, the following cotton cultivars are transformed. Pima, sea Island cotton and upland varities. 21. McCabe DE and Martineli BJ --do-- --do-- --do-- --do-- (1992) Particle mediated transformation of cotton. Patent No. PCT/US92/0172 22. Chlan CA, Lin J, Carry JW & Organogenesis from BA, IAA Embryonic axes Embryonic axes were aseptically Cleveland TE, 1995. A procedure pre-existing meristems removed and bombarded with DNA for biolistic transformation and coated 1.6 A.degree. gold particle at a regeneration of transgenic cotton repture pressure 90 or 110 kg/cm.sup.2. from meristematic tissue. Plant After bombardment, these tissues Mol. Bio. Rep. 13:31-37 were grown in to whole plant in the presence of kannamycin selection. Single axis gave only on plant.